Finding New Proteins Through Protein Expression, Characterization, and Gel Electrophoresis
As new diseases are being discovered, scientists are on the clock to find cures. One of the methods used now a days is protein purification, which is a series of steps to isolate a single type of protein from a complex mixture. The process separates the protein from its non protein parts and at the end separate the desired protein from the other proteins. Strains of E.Coli are used in the purification process to separate proteins for experimental work. There are three steps to protein purification, first the desired protein is overexposed due to the fact there is a decreased amount of the target protein. Then the overexposed protein is lysed so the contents inside the cells are released. Then the insoluable cell components are centrifuged out and the protein is purified using an affinity tag. Lastly the protein is denatured by introducing it to a solution that includes sodium dodecyl sulfate. After the protein is denatured it aquires a negative charge. Then the protein undergoes electrophoresis so researcher can identify if the target protein exists in the solution and whether or not the sample contains other proteins that were not removed by the purification MATERIALS: Protein Expression: Ice bucket, 42oC water bath, gas burner, 2 x 14 ml clear sterile round-bottom tubes, 37oC incubator, coli rollers, 2 LB Agar Amp plates, 1 spare Agar plate without antibiotic, competent cells on ice, plasmid DNA on ice, LB media, SOC media, pipette with pipette tips, gas burner, and Ampicillin stock (50mg/ml).
Protein Purification: Ice bucket, beaker of RT (room temp) water, 1M Imidazole, 10 x PBS, 1.7 ml centrifuge tubes, 2 x 10 ml round bottom tubes, 4 x 15 ml conical tubes, Bio-rad Econochromatography column with yellow cap and clear round top.
Protein Characterization: Mini-PROTEAN electrophoresis tank and lid, power supply and leads, TGS running buffer, Bio-Rad pre-cast polycrylamide gel, 100 ml 6x gel (or sample) loading buffer, protein samples #1-6 from Expression and Purification labs, molecular weight standards, plastic container with lid, and imperial protein stain. METHODS: For the primary experiment, the protein expression, there were three plates. One contained DNA, one did not contain DNA, and another “fun” plate which contained snot. Once the E. coli was obtained, it was thawed and used within five minutes and was placed into 2 different tubes, both were centrifuged. After they were centrifuged the plasmid DNA was added to only the “DNA” tube while none was added to the “control”. These mixtures were then pipetted onto their respective plates. In order to create a larger culture for the target protein, first a starter culture had to be started. Adding a culture from the “DNA” ager plate into a tube that contained both LB media and ampicillin did this. Then these were used to attain a larger batch for the desired protein. By transferring a part of the colony into two earlenmyer flasks and again adding LB media and ampicillin. After incubating the flasks for 6-24 hours in a shaking at 37˚C and 200-350 rpm, the cultures turned purple, which meant there was a good amount of culture in the flask. The cultures were poured into two tubes which were then centrifuged to separate the protein from the non proteins aspects of the liquid. Afterwards, 2.5 ml of 1x PBS was added to the tubes containing the protein along with lysozymes so the protein inside the cells would be released.
The next step to protein purification is to purify the protein. The lysozymes that were added had successfully lysed the cells and expelled the contents within the cell. The tubes were then centrifuged to separate the protein from unwanted cell contents. A syringe filter was used to filter large particulate matter. The proteins were then purified using a using a combination of batch (Ni-NTA resin/buffer mix) and column chromotography. Then the desired protein was filtered out using a solution containing imidazole. The elution was then analyzed on a spectrophotometer at both 280nm and 540nm. Through using beers law the yield was found at both wavelengths, which is the concentration of the two proteins in the solution. The last step of protein purification is protein characterization. In this step the samples collected in the previous two steps were combined with sodium dodecyl sulfate and the samples were pipetted into a Mini-PROTEAN electrophoresis tank containing a precast polyacrylamide gel cassette with TGS 1X buffer. The electrophoresis ran and afterwards the gel was collected and washed three times with nanopure water, then washed with imperial protein stain. The imperial protein stain was discarded and the gel was again washed with nanopure and was left for the night. Lastly the gel was dried in a heated vacuumed presser.
Lab Protein Expression
Figure 1 - An image showing the three agar plates. On the right is the plate containing containing ampicillin and the transformed bacterial specimen NEB pGEM-gbr22. In the middle is the fun group with saliva and no ampicillin. On the right is ampicilllin with the non-recombinant bacterial species NEB. There was no growth in any of the plates.
Figure 2 - An image showing the agar plate with containingampicillin and the transformed bacterial specimen NEB pGEM-gbr22 (leftin Figure 1). Unlike in figure 1, after an estimated nine hours there was growth of a colony in the middle of the ager plate. This showed that the experiment was being done successfully.
Figure 3 - An image showing the two cultures Justin and I grew from the transformed bacteria. Both cultures are the exact same so just in case there is a mistake with one of them, there is a spare.
Lab Protein Purification
(Figure 1) This figure shows both elution 1 (right) and elution 2 (left). As it can be seen there is a color difference between the two. Elution 1 is purple where as elution 2 is clear.
Figure 2 - An image showing the two wet cell pellets isolated from the two flasks in Figure 1. The cell pellets weighed 1.06 g (left) and .87 (right).
(Figure 2a) The absorbance spectrum for gbr22. This was taken at a wavelength of 280nm and the absorbance was found to be 0.364.
The absorbance spectrum for gbr22. This was taken at a wavelength of 280nm and the absorbance was found to be 0.209.
(Figure 3a) The absorbance spectrum for gbr22. This was taken at a wavelength of 574nm (the wavelength for maximal absorption) and the absorbance was found to be 0.72.
(Figure 3b) The absorbance spectrum for gbr22. This was taken at a wavelength of 574nm (the wavelength for maximal absorption) and the absorbance was found to be 0.76.
BEER’S LAW CALCULATION
A = Ebc A is absorbance E is molar absorptivity (with units L mol-1 cm-1) b is path length (with unit cm)
c is concentration (with units mol/L) Yield at the wavelength 280 nm A = .0.2865 E = 38850 b = .100 cm (.0.2865)/(38850 X 0.100 cm) = 7.3745x10^-5 (7.3745x10^-5) (1.00 L) X (1.00 L)/(1.00X10^3 ml) X (5.00 ml) X (25,794.2 g) c = .00951g Yield at the maximum absorbance wavelength 574 nm A = .74 E = 118300 b = 0.100 cm (.740)/(118300 X 0.100 cm) = 6.255X10^-5 M (6.255X10^-5 M)/(1.00 L) X (1.00 L)/(1.00X10^3 ml) X (5.00 ml) X (25,794.2 g) c = .0081g
Lab Protein Characterization
(Figure 1) Above is the gel before it was dried. On the far left is the molecular weight standard then after that its sample 1-6.
(Figure 2) Above is a picture of the gel from electrophoresis after it was dried. The first row on the left is the molecular weight standard, then the next bands are sample 1,2,3,4,5,and 6.
(Figure 3) Picture of the pagerule prestained ladder. This prestained protein MW marker is designed for monitoring the progress of SDS-polyacrylamide gel electrophoresis.
DISCUSSION
The majority of the lab was done, with only few errors here and there. The first part of the lab, the protein expression, had some issues as we did not have any colonies on our “DNA” ager plate. This may have been because the purple gene may not have had enough time to express itself. So we had to borrow another group’s plate. But colonies did begin to sprout after a little bit more incubation. Also the fun plate grew very well in comparison to the “DNA” and “control” plate. The fun plate actually had colonies growing inside. In the protein purification, it can be seen that most of the protein was in elution 1 because elution 1 is more purple than elution 2. The gel electrophoresis part was done successfully but there seems to be some contamination. Contamination probably occurred because of an inadequate wash of the chromatography column before gathering the supposed purified protein. Using figure 3 it was determined that the molecular weight in sample 5 was around 25kDa, which is equal to 4.1535x10^-12 grams. This was then multiplied by (6.022*10^23) to find the molecular weight of the protein, which was 24999.4 g/mol. Since this is very close to the actual molecular weight of the protein, it is safe to say sample 5 contains the target protein. CONCLUSION
These labs were done to show how target proteins can be separated and be used as potential drugs. The steps using protein expression, protein purification, and gel electrophoresis all play a step in finding the correct target protein. The protein gbr22 was targeted in E. coli and was then expressed in this lab. The next step would be to find other proteins to express and purify so they can also be used in other drugs.
2. Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification.Nat Methods2008,5(2), 135-46.
Finding New Proteins Through Protein Expression, Characterization, and Gel Electrophoresis
As new diseases are being discovered, scientists are on the clock to find cures. One of the methods used now a days is protein purification, which is a series of steps to isolate a single type of protein from a complex mixture. The process separates the protein from its non protein parts and at the end separate the desired protein from the other proteins. Strains of E.Coli are used in the purification process to separate proteins for experimental work. There are three steps to protein purification, first the desired protein is overexposed due to the fact there is a decreased amount of the target protein. Then the overexposed protein is lysed so the contents inside the cells are released. Then the insoluable cell components are centrifuged out and the protein is purified using an affinity tag. Lastly the protein is denatured by introducing it to a solution that includes sodium dodecyl sulfate. After the protein is denatured it aquires a negative charge. Then the protein undergoes electrophoresis so researcher can identify if the target protein exists in the solution and whether or not the sample contains other proteins that were not removed by the purification
MATERIALS:
Protein Expression: Ice bucket, 42oC water bath, gas burner, 2 x 14 ml clear sterile round-bottom tubes, 37oC incubator, coli rollers, 2 LB Agar Amp plates, 1 spare Agar plate without antibiotic, competent cells on ice, plasmid DNA on ice, LB media, SOC media, pipette with pipette tips, gas burner, and Ampicillin stock (50mg/ml).
Protein Purification: Ice bucket, beaker of RT (room temp) water, 1M Imidazole, 10 x PBS, 1.7 ml centrifuge tubes, 2 x 10 ml round bottom tubes, 4 x 15 ml conical tubes, Bio-rad Econochromatography column with yellow cap and clear round top.
Protein Characterization: Mini-PROTEAN electrophoresis tank and lid, power supply and leads, TGS running buffer, Bio-Rad pre-cast polycrylamide gel, 100 ml 6x gel (or sample) loading buffer, protein samples #1-6 from Expression and Purification labs, molecular weight standards, plastic container with lid, and imperial protein stain.
METHODS:
For the primary experiment, the protein expression, there were three plates. One contained DNA, one did not contain DNA, and another “fun” plate which contained snot. Once the E. coli was obtained, it was thawed and used within five minutes and was placed into 2 different tubes, both were centrifuged. After they were centrifuged the plasmid DNA was added to only the “DNA” tube while none was added to the “control”. These mixtures were then pipetted onto their respective plates.
In order to create a larger culture for the target protein, first a starter culture had to be started. Adding a culture from the “DNA” ager plate into a tube that contained both LB media and ampicillin did this. Then these were used to attain a larger batch for the desired protein. By transferring a part of the colony into two earlenmyer flasks and again adding LB media and ampicillin. After incubating the flasks for 6-24 hours in a shaking at 37˚C and 200-350 rpm, the cultures turned purple, which meant there was a good amount of culture in the flask. The cultures were poured into two tubes which were then centrifuged to separate the protein from the non proteins aspects of the liquid. Afterwards, 2.5 ml of 1x PBS was added to the tubes containing the protein along with lysozymes so the protein inside the cells would be released.
The next step to protein purification is to purify the protein. The lysozymes that were added had successfully lysed the cells and expelled the contents within the cell. The tubes were then centrifuged to separate the protein from unwanted cell contents. A syringe filter was used to filter large particulate matter. The proteins were then purified using a using a combination of batch (Ni-NTA resin/buffer mix) and column chromotography. Then the desired protein was filtered out using a solution containing imidazole. The elution was then analyzed on a spectrophotometer at both 280nm and 540nm. Through using beers law the yield was found at both wavelengths, which is the concentration of the two proteins in the solution.
The last step of protein purification is protein characterization. In this step the samples collected in the previous two steps were combined with sodium dodecyl sulfate and the samples were pipetted into a Mini-PROTEAN electrophoresis tank containing a precast polyacrylamide gel cassette with TGS 1X buffer. The electrophoresis ran and afterwards the gel was collected and washed three times with nanopure water, then washed with imperial protein stain. The imperial protein stain was discarded and the gel was again washed with nanopure and was left for the night. Lastly the gel was dried in a heated vacuumed presser.
Lab Protein Expression
Lab Protein Purification
BEER’S LAW CALCULATION
A = EbcA is absorbance
E is molar absorptivity (with units L mol-1 cm-1)
b is path length (with unit cm)
c is concentration (with units mol/L)
Yield at the wavelength 280 nm
A = .0.2865
E = 38850
b = .100 cm
(.0.2865)/(38850 X 0.100 cm) = 7.3745x10^-5
(7.3745x10^-5) (1.00 L) X (1.00 L)/(1.00X10^3 ml) X (5.00 ml) X (25,794.2 g)
c = .00951g
Yield at the maximum absorbance wavelength 574 nm
A = .74
E = 118300
b = 0.100 cm
(.740)/(118300 X 0.100 cm) = 6.255X10^-5 M
(6.255X10^-5 M)/(1.00 L) X (1.00 L)/(1.00X10^3 ml) X (5.00 ml) X (25,794.2 g)
c = .0081g
Lab Protein Characterization
DISCUSSION
The majority of the lab was done, with only few errors here and there. The first part of the lab, the protein expression, had some issues as we did not have any colonies on our “DNA” ager plate. This may have been because the purple gene may not have had enough time to express itself. So we had to borrow another group’s plate. But colonies did begin to sprout after a little bit more incubation. Also the fun plate grew very well in comparison to the “DNA” and “control” plate. The fun plate actually had colonies growing inside.In the protein purification, it can be seen that most of the protein was in elution 1 because elution 1 is more purple than elution 2.
The gel electrophoresis part was done successfully but there seems to be some contamination. Contamination probably occurred because of an inadequate wash of the chromatography column before gathering the supposed purified protein. Using figure 3 it was determined that the molecular weight in sample 5 was around 25kDa, which is equal to 4.1535x10^-12 grams. This was then multiplied by (6.022*10^23) to find the molecular weight of the protein, which was 24999.4 g/mol. Since this is very close to the actual molecular weight of the protein, it is safe to say sample 5 contains the target protein.
CONCLUSION
These labs were done to show how target proteins can be separated and be used as potential drugs. The steps using protein expression, protein purification, and gel electrophoresis all play a step in finding the correct target protein. The protein gbr22 was targeted in E. coli and was then expressed in this lab. The next step would be to find other proteins to express and purify so they can also be used in other drugs.
REFERENCES
1. Protein Expression and Purification Core Facility. http://www.embl.de/pepcore/pepcore_services/index.html (accessed April 14).
2. Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification. Nat Methods 2008, 5 (2), 135-46.